Journal of Pharmaceutical and Biomedical Analysis

26 (2001) 291 299

www.elsevier.com/locate/jpba

Simultaneous determination of malathion, permethrin,

DEET (N,N-diethyl-m-toluamide), and their metabolites in
rat plasma and urine using high performance liquid
chromatography
Aqel W. Abu-Qare, Mohamed B. Abou-Donia *
Department of Pharmacology and Cancer Biology, Duke Uni6ersity Medical Center, PO Box 3813, Durham, NC 27710 USA

Received 31 October 2000; received in revised form 30 January 2001; accepted 9 February 2001

Abstract

A method was developed for the separation and quantification of the insecticide malathion (O,O-dimethyl-S-(1,2-
carbethoxyethyl) phosphorodithioate), its metabolite malaoxon (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphoroth-
ioate), the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxy-
phenyl)methylester), two of its metabolites m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, the insect repellent
N,N-diethyl-m-toluamide (DEET), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The
method used high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection
at 210 nm. The compounds were separated using gradient of 45 99% acetonitrile in water (pH 3.5) at a flow rate
ranging between 0.5 and 2 ml/min in a period of 15 min. The retention times ranged from 7.4 to 12.3 min. The limits
of detection ranged between 20 and 100 ng/ml, while limits of quantitation were 50 150 ng/ml. Average percentage
recovery of five spiked plasma samples were 80.1 9 4.2, 75.2 9 4.6, 84.5 9 4.0, 84.3 9 3.4, 82.8 93.9, 83.9 95.5,
82.29 6.0, 83.1 9 4.3, and from urine 78.8 93.9, 76.4 9 4.9, 82.3 9 4.5, 82.5 9 3.9, 81.4 9 4.0, 83.9 94.3, 81.5 95.0,
and 84.5 9 3.8 for, malathion, malaoxon, DEET, m-toluamide, m-toluic acid, permethrin, m-phenoxybenzyl alcohol,
and m-phenoxybenzoic acid, respectively. The method was reproducible and linear over range between 100 and 1000
ng/ml. This method was applied to analyze the above chemicals and metabolites following combined dermal
administration in rats. 2001 Elsevier Science B.V. All rights reserved.

Keywords: West Nile Virus; Reversed phase HPLC; Combined exposure; Malathion; DEET; Permethrin

1. Introduction

Malathion is a widely used organophosphate

* Corresponding author. Tel.: + 1-919-6842221; fax: + 1- insecticide in agriculture [1] and in public health
919-6818224.
E-mail address: donia@acpub.duke.edu (M.B. Abou-Do- programs for controlling mosquito born-disease
nia). [2]. Malathion disrupts the nervous system by

0731-7085/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 7 3 1 - 7 0 8 5 ( 0 1 ) 0 0 4 0 7 - 1
292 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299
inhibiting cholinesterase enzymes [3]. Toxic symp-
toms resulting from human exposure to malathion,
include breathing problems, headache, nausea and
dizziness, while high exposure can produce fatal
poisoning [1,3]. Furthermore, malathion, has found
to cause oxidative stress in rats and mice [4,5], and
it is a suspected hormone disrupter [6]. Permethrin
is a pyrethroide insecticide applied inside homes
and in public places [7]. Pyrethroides modify
sodium channel to open longer during a depolariza-
tion pulse [8], and act as a weak hormone mimic
in test tube studies [9]. N,N-diethyl-m-toluamide
(DEET) was applied as insect repellent on the skin
against mosquitoes and other biting insects [10].
DEET had direct effect on the nervous system in
laboratory animals resulting in spongiform
myelinopathy in the brain stem with signs include
ataxia, seizures, and death [11]. In other study,
extensive and repeated topical application of
DEET resulted in human poisoning including two
deaths [12].
Absorption, disposition, metabolism, and excre-
tion of malathion has been studied in animals and
humans [3,4,13]. Permethrin has been reported to
be absorbed into plasma, metabolized, and ex-
creted as metabolites in the urine following oral or
intravenous dose in rats [14], and in rabbits [15].
Absorption and excretion of DEET and metabo-
lites were rapid after dermal application in human
[16,17], in rats [18], and in dogs [19].
Several analytical methods have been used for
identification and quantification of the above
chemicals and their metabolites, when applied as
individual, in plasma and urine samples. These
methods used high performance liquid chromatog-
raphy (HPLC) [14 25], high performance liquid
chromatography-mass spectrometry (HPLC-MS)
[27], gas chromatography (GC) [15,28,29], gas
chromatography-mass spectrometry (GC-MS) [26],
and high performance thin-layer chromatography
(HPTLC) [30].
Recently malathion, permethrin and DEET have
been simultaneously used to protect against West
Nile Virus by killing adult mosquitoes in some parts
of the United States [9,31]. As a result, thousands
of people could be exposed to malalthion, perme-
thrin and DEET inside homes and in public places.
To examine their possible pharmacokinetics inter-
actions, we developed a reliable method for simul-
taneous analysis of malathion, permethrin, DEET
and their metabolites in rat plasma and urine using
solid phase extraction coupled with reversed phase-
HPLC.
2. Experimental
2.1. Chemicals and materials
Malathion (99% O,O-dimethyl-S-(1,2-car-
bethoxyethyl) phosphorodithioate), malaoxon
(98% 0,0 -dimethyl-S-(1,2-carbethoxyethyl) phos-
phorothioate), m-phenoxybenzoic acid, and m-
phenoxybenzyl alcohol (Fig. 1) were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). Per-
methrin (99% 3-(2,2-dichloro-ethenyl)-2,2-dimeth-
ylcyclopropanecarboxylicacid(3-phenoxyphenyl)
methylester) was obtained from Chem Service, Inc.
(West Chester, PA, USA). DEET (98% N,N-Di-
ethyl-m-toluamide) (Fig. 1) was obtained from
Aldrich Chem Co., Inc. (Milwakee, WI, USA).
m-Toluamide and m-toluic acid were purchased
from Fisher Scientific (Pittsburgh, PA, USA). Wa-
ter (HPLC grade) and acetonitrile were obtained
from Mallinckrodt Baker, Inc. (Paris, Kentucky,
USA). C18 Sep-Pak cartridges were obtained from
Waters Corporation (Waters Corporation, Mil-
ford, MA, USA).
2.2. Animals
Rats (SpragueDawley) were purchased from
Zivic Miller (Zelienople, PA, USA). The animals
were kept in plastic metabolic cages. Five rats were
treated with a combined dermal dose of 10 mg/kg
of malathion, a 200 mg/mg of DEET, and a 1.3
mg/kg of permethrin. The doses were selected to
represent real-life exposure: DEET and permethrin
doses were determined by US Department of De-
fense (personal communication), while the dose of
malathion is approximately 1% of its dermal LD50
in rats. Five untreated control rats were treated
with dermal dose of ethanol. The animals were held
in metabolic cages as to allow collection of urine
samples. Urine samples were collected from treated
and controls 12 h after dosing. The animals were
 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299
anesthetized with halothane and sacrified by heart
exsanguinations at 12 h, blood was collected via
heart puncture with a heparinized syringe and
centrifuged at 2400 rpm for 15 min at 5C to
separate plasma. Urine and plasma samples were
stored at 20C prior to analysis.
2.3. Instrumentation
The liquid chromatographic system (Waters
2690 Separation Module), consisted of a Waters
600E Multisolvent delivery system pumps, a Wa-
ters Ultra WISP 715 autoinjector, and a Waters
2487 Dual l absorbance detector (Waters Corpo-
ration, Milford, MA). A guard column (Supelco,
2 cm 4.0 mm, 5mm (Supelco Park, Bellefonte,
PA), and a reversed-phase C18 column
mBondapak C18 125A, 10 mm, 3.9 300 mm
were used, (Waters Corporation, Milford, MA).
2.4. Sample preparation
A 0.5 ml plasma and urine samples from un-
treated rats were spiked with concentrations rang-
Fig. 1. Structures of malathion, malaoxon, permethrin, m-phenoxybenzyl alcohol, m-phenoxybenzoic acid, DEET, m-toluamide,
and m-toluic acid.
293
ing between 100 and 1000 ng/ml of each of
malathion, malaoxon, permethrin, m-phenoxy-
benzyl alcohol, m-phenoxybenzoic acid, DEET,
m-toluamide, and m-toluic acid. Spiked and
treated samples were acidified with 1 N acetic acid
(pH 3.5). Disposable C18 Sep-Pak Vac 3cc (500
mg) cartridges (Waters Corporation, Milford,
MA) were conditioned with 3 ml of acetonitrile,
then equilibrated using 3 ml of water prior use.
The spiked urine and plasma samples were vor-
texed for 30 s, centrifuged for 5 min at 1000g,
and the supernatant was loaded into the dispos-
able cartridges, then washed with 3 ml of water,
and eluted two times by 2 ml of acetonitrile, then
by 2 ml of methanol, and reduced to 500 ml using
stream of nitrogen, prior to analysis by HPLC.
2.5. Chromatographic conditions
A 10 ml solution of plasma or urine solutions
was injected into HPLC. The mobile phase was
water (adjusted to pH 3.50 using 1 N acetic acid):
acetonitrile gradient at flow rate programmed
from 0.5 ml/min from zero-9 min, increased to 2
294 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299
ml/min by 10 min, then returned to 0.5 ml/min at
13 min. The gradient started at 45% acetonitrile
until 9 min, increased to 90% acetonitrile by 10 min.
Then the system returned to 45% acetonitrile at 13
min where it was kept under this condition for 2
min to re-equilibrate. The eluents were monitored
by UV detection of wavelength of 210 nm. The
chromatographic analysis was performed at ambi-
ent temperature.
2.6. Calibration procedures
Five different calibration standards of a mixture
of malathion, malaoxon, permethrin, m-phenoxy-
benzyl alcohol, m-phenoxybenzoic acid, DEET,
m-toluamide, and m-toluic acid were prepared in
acetonitrile. Their concentrations ranged from 100
to 1000 ng/ml. Linear calibration curves were
obtained by plotting the peak areas of the individ-
ual chemicals as a function of the concentration
using GraphPad Prism program for windows
(GraphPad Software, Inc., San Diego, CA, USA).
The standard curves were used to determine recov-
ery of the chemicals from plasma and urine sam-
ples.
2.7. Limits of detection (LOD) and limits of
quantitation (LOQ)
Limits of detection and quantitation were deter-
mined at the lowest concentration to be detected or
quantify, taking into consideration a 1:3 and 1:10
baseline noise: calibration point ratio, respectively.
The LOQ was repeated five times for confirmation.
3. Results
3.1. Standard calibration cur6es
The standard calibration curves of peak area
against concentration of malathion, malaoxon,
permethrin, m-phenoxybenzyl alcohol, m-phe-
noxybenzoic acid, DEET, m-toluamide, and m-
toluic acid are shown in Fig. 2. Linearity of the
calibration curves for the three compounds was
achieved at concentrations ranging from 100 to
1000 ng/ml.
3.2. Chromatogram
Chromatographic profiles were obtained for rat
plasma and urine samples after solid phase extrac-
tion using C18 Sep Pak cartridges under HPLC
conditions as described above (Fig. 3 Fig. 4).
Retention times were 7.4, 8.3, 9.1, 9.7, 10.2, 10.7,
11.4 and 12.3 min for m-toluamide, m-toluic acid,
malaoxon, DEET, m-phenoxybenzyl alcohol, m-
phenoxybenzoic acid, malathion, and permethrin,
respectively. The total run time was 15 min. Clean
chromatogram shows no interference from endoge-
nous substances in plasma and urine samples and
proves the selectivity of the method.
3.3. Extraction efficiency and reco6ery
The average extraction recoveries of malathion,
malaoxon, permethrin, m-phenoxybenzyl alcohol,
m-phenoxybenzoic acid, DEET, m-toluamide, and
m-toluic acid were determined at concentrations
ranging between 100 and 1000 ng/ml (Table 1 Table
2). Spiked plasma and urine samples were extracted
and analyzed for each concentration in five repli-
cates. Average percentage recovery of five spiked
plasma samples were 80.19 4.2, 75.29 4.6, 84.59
4.0, 84.39 3.4, 82.893.9, 83.99 5.5, 82.296.0,
83.19 4.3, and from urine 78.893.9, 76.494.9,
82.39 4.5, 82.59 3.9, 81.494.0, 83.994.3,
81.59 5.0, and 84.59 3.8 for, malathion,
malaoxon, DEET, m-toluamide, and m-toluic acid,
permethrin, m-phenoxybenzyl alcohol, and m-phe-
noxybenzoic acid, respectively.
3.4. Limits of detection
Blank plasma and urine samples from untreated
rats were used as references for plasma and urine
collections. Limits of detection were calculated
from a peak signal to noise ratio of 3:1. The
resulting detection limits were 50, 100, 50, 20, 20,
50, 50, and 50 ng/ml for malathion, malaoxon,
permethrin, m-phenoxybenzyl alcohol, m-phe-
noxybenzoic acid, DEET, m-toluamide, and m-
toluic acid, respectively.
3.5. Limits of quantitation (LOQ)
Limits of quantitation in control plasma sam-
 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299 295

Fig. 2. Standard calibration curves of malathion, malaoxon, permethrin, m-phenoxybenzyl alcohol, m-phenoxybenzoic acid, DEET,
m-toluamide, and m-toluic acid.

ples were determined to be 100, 150, 100, 50, 50,
100, 100, and 100 ng/ml, while from spiked urine
samples were 100, 150, 150, 100, 100, 100, 100,
and 100 ng/ml for malathion, malaoxon, per-
methrin, m-phenoxybenzyl alcohol, m-phenoxy-
benzoic acid, DEET, m-toluamide, and m-toluic
acid, respectively.
3.6. Application of the method to biological samples
In order to test its applicability, the method
was applied for the analysis of the three chemi-
cals and their metabolites following combined
dermal administration in rats. The rats were sac-
rificed at 12 h following dosing. In plasma,
malathion, DEET, m-toluamide, permethrin,
and m-phenoxybenzyl alcohol were detected.
Their levels were 2869 53, 7029 186, 273994,
1869 32, and 2129 38 ng/ml, respectively. Also,
traces of malaoxon were detected in plasma
samples. In urine, DEET, and m-phenoxybenzyl
alcohol have been detected 12 h after treatment.
Their levels were 4259 142 and 1839 36 ng/ml,
respectively. The results were corrected based on
the percentage recoveries of the above chemicals
from plasma and urine samples.
296 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299

4. Discussion Linearity of standard calibration curves for the

In this study, we developed an HPLC method
for the separation and quantification of malalth-
ion, permethrin, DEET and their metabolites in
rat plasma and urine. The method is significant at
this time where the three chemicals are in use
against West Nile Virus, and possible interactions
between the three chemicals could lead to more
toxicity. The method could be used in studying
pharmacokinetic interactions between the com-
pounds. The chromatogram obtained following
solid phase extraction and HPLC analysis shows
no interference from plasma and urine endoge-
nous substances, indicating an efficient clean up
method used and proving selectivity of the
method. Simultaneous determination of the par-
ent compounds and their metabolites are cost
efficient and save time for sample preparation and
clean up.
chemicals in this method was obtained over a
range between 100 and 1000 ng/ml. This range is
in agreement with earlier studies using similar
ranges. Eilln et al. [20] reported linear range be-
tween 40 and 500 ng/ml for DEET in plasma
using HPLC, while Taylor et al. [32] reported
linearity over a range between 19 and 1910 ng/ml
for DEET using gas chromatography.
Recoveries of the anlaytes were suitable for
application of the method for the determination
of treated samples for parent compounds and
their metabolites following real-life exposure. The
selected dose of malathion represent less than 1%
of its dermal LD50 in rats [1], while the dose of
permethrin and DEET was determined by US
Department of Defense (personal communica-
tions). Recoveries of the analytes in this method
were between 75 and 91%. This range lies within

Fig. 3. Chromatogram of spiked plasma sample with 500 ng/ml of (A) m-toluamide, (B) m-toluic acid, (C) malaoxon, (D) DEET,
(E) m-phenoxybenzyl alcohol, (F) m-phenoxybenzoic acid, (G) malathion, and (H) permethrin under established HPLC conditions.
 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299 297

Fig. 4. Chromatogram of spiked urine sample with 500 ng/ml of (A) m-toluamide, (B) m-toluic acid, (C) malaoxon, (D) DEET, (E)
m-phenoxybenzyl alcohol, (F) m-phenoxybenzoic acid, (G) malathion, and (H) permethrin under established HPLC conditions.

Table 1
Percentage (%) recoverya of malathion, DEET, permethrin, and their metabolites from rat plasma

Concentration Malathion Malaoxon DEET m-Toluamide m-Toluic Permethrin m-Phenoxybe m-Phenoxyben

(ng/ml) acid nzyl alcohol zoic acid

1000 83.294.6 79.29 3.8 91.1 9 2.7 88.7 9 4.2 88.5 9 3.8 85.7 9 5.8 84.9 95.1 88.2 92.6
500 83.993.0 78.094.0 84.2 95.2 86.3 92.8 85.9 9 2.8 88.3 9 5.2 82.3 94.8 84.2 94.9
400 80.5 9 5.7 76.1 92.9 83.7 94.2 85.1 94.3 82.6 9 3.2 85.2 9 4.9 84.7 98.3 82.9 95.6
200 78.294.8 70.19 6.9 82.9 9 3.8 81.5 93.9 80.7 94.3 81.9 9 5.6 82.1 96.2 83.7 9 4.7
100 75.293.1 72.4 94.7 80.5 94.2 79.8 91.9 76.5 95.2 78.2 9 6.2 77.1 95.7 76.5 9 3.8

a
Values are expressed as mean 9 S.D. of five replicates.

the reported values in the literature, taking into
consideration simultaneous determination of the
three chemicals and their metabolites. Recoveries
of DEET from serum and urine were reported to
be 9395%, and 65 70%, respectively, using GC-
MS as an analytical technique [33], while recovery
of DEET from water samples was 45.6% using
Micllar kinetic chromatography method [34]. In
earlier studies, recovery of pyrethroids and
metabolites from rat urine ranged between 90 and
98% using GC-MS [26]. Bissacot and Vassilieff
[35] reported recoveries between 78 and 91% of
four pyrethroids from milk and blood of lactating
dairy cows using HPLC. Futagami et al. [30]
298 A.W. Abu-Qare, M.B. Abou-Donia / J. Pharm. Biomed. Anal. 26 (2001) 291299

Table 2
a
Percentage (%) recovery of malathion, DEET, permethrin, and their metabolites from rat urine

Concentration Malathion Malaoxon DEET m-Toluamide m-Toluic Permethrin m-Phenoxybe m-Phenoxyben

(ng/ml) acid nzyl alcohol zoic acid

1000 83.09 5.1 78.09 4.8 85.2 93.8 90.1 93.0 82.1 92.8 85.3 94.8 83.1 9 4.9 90.1 93.6
500 81.99 2.0 75.393.8 82.0 96.1 84.7 93.6 83.1 93.7 87.0 94.2 80.6 9 5.4 87.2 94.2
400 77.29 3.7 79.595.9 80.3 95.6 80.2 95.1 83.2 94.0 88.2 93.9 84.3 9 6.1 82.6 94.9
200 76.19 4.8 74.396.5 83.2 94.2 78.1 94.1 78.2 9 3.2 80.1 9 3.6 81.1 9 4.0 83.5 93.7
100 76.09 4.1 74.993.7 80.6 92.9 79.2 93.6 80.5 9 6.3 78.3 9 5.2 76.2 9 4.7 79.1 92.8

a
Values are expressed as mean 9S.D. of five replicates.

reported a recovery of 84.5 and 95.8% for
malathion from urine samples using solid phase
and liquid-liquid extraction, respectively,
Malathion recovery from blood sample was be-
tween 85 and 97% [22].
The limits of detection reported in the described
method allow to determination of samples from
treated animals following doses resemble real-life
exposure. The ability to detect parent compounds
and metabolites in plasma after 12 h of dosing is
an evidence of the method suitability. Only traces
of malaoxon have been detected in plasma. This
could be due to its rapid degradation and elimina-
tion. The failure to detect DEET metabolites m-
toluamide and m-toluic acid, permethrin and its
metabolite m-phenoxybenzoic acid in the urine
might due to rapid hydrolysis and conjugation of
permethrin, DEET and the targeted metabolites.
The reported limits of detection in the literature
are consistent with our results for the simulta-
neous determination of the analytes that ranged
between 20 and 100 ng/ml. In earlier study, limits
of detection of malathion using HPTLC were 120
ng/ml [30]. Detection limits of permethrin in urine
samples were 0.3 0.5 mg/l using GC-MS tech-
nique [26]. Bissacot and Vassilieff [35] reported
detection limit of 1 mg/g of four pyrethroids in
milk and blood of lactating dairy cows using
HPLC. The detection limit of DEET was 90
ng/ml and 90 ng/g from urine and serum, respec-
tively, using HPLC-UV method [16], and 15 ng/
ml for DEET in human and dog plasma using
HPLC [36].
A reliable, rapid and simple HPLC method was
developed for separation and quantification of
malathion, permethrin and selected metabolites in
rat spiked and treated plasma and urine samples.
Solid phase extraction was used which selectively
extracted the above chemicals from plasma and
urine samples without interference of an expected
mixture of metabolites and endogenous com-
pounds. The method could be applied routinely
for monitoring of these compounds in human
plasma and urine samples of people exposed to
the compounds in some areas where these chemi-
cals are used to control West Nile Virus. Also this
method could be used to study the pharmacoki-
netic profiles of these compounds, alone and in
combination.
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